Unlike B and T cells, NK cells do not express antigen-specific receptors, yet they can eliminate virus-infected cells and cancer cells without harming normal cells. An important component that provides specificity in target cell recognition is inhibition of NK cells by inhibitory receptors, which recognize surface molecules called major histocompatibility complex (MHC) class I. MHC-specific recognition by inhibitory receptors on NK cells prevents the killing of normal, healthy cells. The major goal of this project is to elucidate the mechanism by which inhibitory receptors block NK cell activation. Natural cytotoxicity is achieved by polarized release of perforin and granzymes at the NKtarget cell immunological synapse. Signal transduction pathways that lead to granule polarization and degranulation are uncoupled in NK cells. Therefore, we were interested in testing whether polarization and degranulation would have the same degree of sensitivity to inhibitory receptors. Expression of human MHC class I molecules, either HLA-C or HLA-E, on a class I-negative cell line blocked granule polarization, degranulation, and CD16-dependent cytokine secretion by NK cell clones that expressed inhibitory receptors of matching HLA specificity. To test inhibition of signals for polarization and degranulation separately, Drosophila S2 cells expressing ICAM-1 with either HLA-C or HLA-E were used. ICAM-1 is a ligand for the beta2 integrin LFA-1, which signals for granule polarization in NK cells. CD16-dependent degranulation and cytokine secretion were not fully inhibited, suggesting that other receptorligand interactions, which occur with 721.221 cells, contribute to inhibition. In contrast, HLA-C or HLA-E on S2 cells co-expressing ICAM-1 were sufficient to block granule polarization induced by either LFA-1, even during concomitant CD16-dependent degranulation. Therefore, granule polarization, rather than degranulation, is the preferred target of inhibitory receptors in NK cells. The main conclusion is that inhibitory receptors are better equipped to stop granule polarization than to block GrzB and chemokine release. Persistent degranulation during inhibition of polarization in IL-2-activated NK cells suggests that it may occur in high inflammatory conditions. Prevention of NK cell cytotoxicity would be better achieved through inhibition of degranulation rather than polarization. However, the possibility of releasing the block in degranulation while maintaining inhibition of polarization endows NK cells with the potential to provide bystander killing while still refraining from direct attacks on MHC class I-positive cells. Control of natural cytotoxicity by MHC class I-specific inhibitory receptors involves recruitment of the tyrosine phosphatase SHP-1, which dephosphorylates the guanine exchange factor Vav. As Vav is essential for proper actin remodeling and synapse formation, Vav inactivation through dephosphorylation provides an efficient way to block NK cell cytotoxicity. However, we have shown recently that the inhibitory signaling pathway is more complex and involves a second component, which relies on a tyrosine phosphorylation step. The small adapter Crk is phosphorylated during inhibition by MHC class I-specific receptors. Crk phosphorylation results in its dissociation from actin cytoskeleton-associated signaling complexes. We then wanted to test whether Crk contributes to signaling by activation receptors, which could explain why Crk may be a target of inhibitory signal transduction pathways. Using primary NK cells over lipid bilayers carrying IgG1 Fc, a ligand for CD16, we have shown that Crk is required for clustering of the CD16 ligand Fc, and for phosphorylation of Vav. HLA-E, the ligand of inhibitory receptor CD94-NKG2A, on the lipid bilayer was sufficient to induce Crk phosphorylation, implying that inhibitory receptors can transmit a signal independently of signals from any other receptor. Addition of HLA-E on the lipid bilayers, in combination with the CD16 ligand Fc, resulted in a reduced number of peripheral Fc microclusters, but promoted central accumulation of Fc and phosphorylation of Crk at inhibitory synapses. Crk was also required for the ability of inhibitory receptors to clear F-actin from the center of synapses. Our data show that inhibitory receptors use Crk as a tool to block NK cell activation, and reveal a dual role of Crk in controlling activation and inhibition of NK cells.